Folylpolyglutamate synthetase (FPGS) catalyses the addition of multiple moles of glutamic acid to the naturally-occurring folate cofactors and is essential for the survival of proliferating mammalian cells, normal and neoplastic. The same enzyme adds up to seven moles of glutamic acid to any of the "classical" folate antimetabolite, including methotrexate, 5,10-dideazatetrahydrofolate, and the thymidylate synthase inhibitor Z-D- 1694. The polyglutamation process is essential for the retention of the naturally-occurring folate cofactors in mammalian cells, but this same process serves to metabolically trap cytotoxic folate antimetabolite in tumor cells. Recent studies have shown that the expression of FPGS is elevated in some types of tumors and that the characteristics of the enzyme seems to be different in some tumors compared with normal adult stem cells and differentiated tissues. This grant proposes to intensively study the characteristics of the active site of the FPGS expressed in mouse and human tumor cells using cellular, biochemical, molecular and structural approaches. These studies will seek to find structural differences between the active site of enzyme expressed in tumor cells and that in normal tissues. If such differences are found, the several newly synthesized groups of folate antimetabolities will be used as a data base to find molecules that are activated to polyglutamates by the one enzyme but not the other. If activation occurs only by tumor enzyme, then a compound cytotoxic, upon polyglutamatin, to some other folate-dependent process will be sought. If binding can be attained by the FPGS expressed by normal stem cells but not by that in tumor, then we shall investigate methods to develop inhibitors of FPGS in normal tissues. Such an approach would allow coadministration of the inhibitor and any of several cytotoxic chemotherapies that rely on polyglutamation for activity, e.g., 5-fluorouracil coadministered with folinic acid, or either dideazatetrahydrofolate or ZD-1694. The utility of folate antimetabolities for the treatment of leukemias and breast carcinomas makes the search for selectivity in the activation of folate antimetabolities one of the most attractive means of rationally directing cytotoxic cancer chemotherapy.